Azodipyrroles of unconjugated and conjugated bilirubin using diazotized ethyl anthranilate in dimethyl sulfoxide

We have developed a diazotization technique in which both conjugated and unconjugated bilirubin react completely. The method represents a crucial modification of the ethyl anthranilate diazo reaction originally described by K. P. M. Heirwegh, J. Fevery, J. A. T. P. Meuwissen, and J. de Groote (1974, Methods Biochem. Anal.22, 205–250). In the presence of dimethyl sulfoxide (2 ml/ml of sample and diazo reagent), conjugated and unconjugated bilirubin in human serum and human, rat, and mouse bile reacted rapidly and completely. The azopigments were stable for at least 4 h. Addition of human serum to unconjugated bilirubin, bilirubin monoglucuronide, and human bile did not influence azopigment formation. Because the reaction solution was optically clear, total azopigments could be measured by spectrophotometry or separated and quantitated by high-performance liquid chromatography without prior extraction into nonpolar solvents. Alternatively, the pigments could also be extracted into 2-pentanone for analysis by thin-layer or high-performance liquid chromatography. This method allows the quantitation of total bilirubin and analysis of individual ethyl anthranilate azopigments after a single diazotization step.     

Uterine and placental 5-HT profile in different gestational period of albino rats

Possible role of 5-HT in pregnancy was investigated in albino rats by biological estimation of uterine and placental 5-HT contents in different periods of gestation in normal and drug treated rats. Uterine 5-HT level increased steadily from day-1 of gestation to reach the peak on day-7; thereafter, the level continued to decline throughout the period till day-20 when 5-HT level was lowest. From day-20, a mild secondary rise started and remained persistent even after parturition. The results show that a critical level of 5-HT in early gestational period is necessary for conception. Manipulation of endogenous 5-HT do not influence duration of gestation.     

Early embryonic development of the vulnerable Shalyni barb,Pethia shalynius (Yazdani & Talukdar, 1975)

The present study provides the first detailed early embryonic development of the Shalyni barb, Pethia shalynius (Yazdani & Talukdar, 1975), a vulnerable cyprinid fish occurring in streams and lentic waters of Meghalaya, northeast India. Induced spawning by synthetic hormone injection in May 2019 was conducted to a pair of mature female and male P. shalynius under controlled conditions in a well-aerated aquarium. Fertilized eggs were spherical, 0.75–0.80 mm (approx.) in diameter, transparent, unpigmented and non-adhesive. A total of 22 developmental stages could be categorized under seven broad periods, viz. the zygote, cleavage, blastula, gastrula, segmentation, pharyngula and hatchling. The first cleavage occurred at 15 min post fertilization (mpf), followed by blastulation at 01:23 hr post-fertilization (hpf), gastrulation at 04:20 hpf, initial somite formation at 07:00 hpf, and pharyngula period at 19:20 hpf, respectively. Embryos hatched between 26–27 hpf and the newly-hatched larvae ranged 2.2–2.5 mm in total length. For naturally-declining populations of this vulnerable fish species, inferences drawn from the present study will help provide a baseline data for its conservation and management, and aid the research fields of developmental biology, biotechnology, molecular biology as well as taxonomy of this species.     

A detailed model and Monte Carlo simulation for predicting DIP genome length distribution in baculovirus infection of insect cells

Baculoviruses have enormous potential for use as biopesticides to control insect pest populations without the adverse environmental effects posed by the widespread use of chemical pesticides. However, continuous baculovirus production is susceptible to DNA mutation and the subsequent production of defective interfering particles (DIPs). The amount of DIPs produced and their genome length distribution are of great interest not only for baculoviruses but for many other DNA and RNA viruses. In this study, we elucidate this aspect of virus replication using baculovirus as an example system and both experimental and modeling studies. The existing mathematical models for the virus replication process consider DIPs as a lumped quantity and do not consider the genome length distribution of the DIPs. In this study, a detailed population balance model for the cell‐virus culture is presented, which predicts the genome length distribution of the DIP population along with their relative proportion. The model is simulated using the kinetic Monte Carlo algorithm, and the results agree well with the experimental results. Using this model, a practical strategy to maintain the DIP fraction to near to its maximum and minimum limits has been demonstrated.     

Phosphonoformate Esters of Anti-HIV Nucleosides: 3′-Azido-3′-deoxythymidine and 2′,3′-Dideoxycytidine Derivatives Containing a Small 5′-(0-Alkoxycarbon-ylphosphinyl) or 5′-(O-Cholesterylcarbonylphosphinyl) Substituent

Abstract

A convenient general method of synthesis of 5′-O-(alkoxycarbonyl)phosphonate esters of 2′,3′-dideoxyribonucleosides is presented, using the 5′-O-(methoxycarbonyl)phosphinyl, 5′-0-(ethoxycarbonyl)phosphinyl, and 5′-O-(cholesterylcarbonyl)phosphinyl derivatives of 3′-azido-3′-deoxythymidine (AZT) and the 5′-0-(ethoxycarbonyl)phosphinyl derivative of 2′,3′-dideoxycytidine (ddC) as examples. Reaction of trimethyl phosphonoformate, methyl phosphonoformate, or dimethyl cholesterylcarbonylphosphonate with phosphorus pentachloride in carbon tetrachloride, followed by direct condensation of the resulting phosphonyl chloride with the nucleoside, gave the fully esterified phosphonoformate derivatives, which on treatment with sodium iodide in tetrahydrofuran underwent selective cleavage of the P-OMe or P-OEt groups, leaving the carboxylate esters intact. The resulting products were converted from sodium salts to ammonium salts by ion-exchange chromatography.     

New facets of larger Nest motifs in proteins

The Nest is a concave-shaped structural motif in proteins formed by consecutive enantiomeric left-handed (L) and right-handed (R) helical conformation of the backbone. This important motif subsumes many turn and helix capping structures and binds electron-rich ligands. Simple Nests are either RL or LR. Larger Nests (>2 residues long) may be RLR, LRL, RLRL, and so forth, being considered as composed of overlapping simple Nests. The larger Nests remain under-explored despite their widely known contributions to protein function. In our study, we address whether the recurrence of enantiomeric geometry in the larger Nests constrains the peptide backbone such that distinct compositional and conformational preferences are seen compared to simple Nests. Our analysis reveals the critical role of the L helical torsion angle in the formation of larger Nests. This can be observed through the higher propensity of residue or secondary structure combinations in LR and LRL backbone conformation in comparison to RL or RLR, although LR/LRL is considerably lower by occurrence. We also find that the most abundant doublets and triplets in Nests have a propensity for particular secondary structures, suggesting a strong sequence-structure relationship in the larger Nest. Overall, our analysis corroborates distinct features of simple and the larger Nests. Such insights would be helpful towards in-vitro design of peptides and peptidomimetic studies.     

Circular Dichroism Studies of the Structure of DNA Complex with Berberine

Abstract

The binding of the benzodioxolo-benzoquinolizine alkaloid, berberine chloride to natural and synthetic DNAs has been studied by intrinsic and extrinsic circular dichroic measurements. Binding of berberine causes changes in the circular dichroism spectrum of DNA as shown by the increase of molar ellipticity of the 270nm band, but with very little change of the 240nm band. The molar ellipticity at the saturation depends strongly on the base composition of DNA and also on salt concentration, but always larger for the AT rich DNA than the GC rich DNA The features in the circular dichroic spectral changes of berberine-synthetic DNA complexes were similar to that of native DNA but depends on the sequence of base pairs.

On binding to DNA and polynucleotides, the alkaloid becomes optically active. The extrinsic circular dichroism developed in the visible absorption region (300–500nm) for the berberine-DNA complexes shows two broad spectral bands in the regions 425–440nm and 340–360nm with the maximum varying depending on base composition and sequence of DNA While the 425nm band shows less variation on the binding ratio, the 360nm band is remarkably dependent on the DNA/alkaloid ratio. The generation of the alkaloid associated extrinsic circular dichroic bands is not dependent on the base composition or sequence of base pairs, but the nature and magnitude of the bands are very much dependent on these two factors and also on the salt concentration. The interpretation of the results with respect to the modes of the alkaloid binding to DNA are presented.